Leptin, the product of the ob gene, is a key player in energy homeostasis and body weight control. It is a 16-kDa circulating protein with a structure resembling 4-α-helical bundle cytokines (Madej et al., 1995). It is mainly secreted by adipose cells, and the circulating level of this hormone strongly correlates with white adipose tissue mass. Leptin regulates energy expenditure and food intake by activating its receptor in certain nuclei of the hypothalamus (Halaas et al., 1995; Campfield et al., 1995; Pelleymounter et al., 1995). Loss-of-function mutations within the genes for leptin (Montague et al., 1997), or for its receptor (Lee et al., 1997; Chen et al., 1996; Clement et al., 1998) cause complex syndromes characterized by morbid obesity, hyperglycemia, hyperinsulinemia, and reduced fertility. Numerous data suggest that leptin also has direct effects on tissues outside the brain, which may help explain its role on basal metabolism, reproduction, hematopoiesis and regulation of the immune response (Chebab et al., 2002; Baile et al., 2000; Fantuzzi and Faggioni, 2000; Matarese et al., 2002).
The leptin receptor (LR) is composed of a single subunit, encoded by the db gene (Lee et al., 1996; Chen et al., 1996; Tartaglia et al., 1995; Cioffi et al., 1996), and which is a member of the class I cytokine receptor family. It contains two so-called CRH modules, which are formed by two barrel-like domains, each approximately 100 amino acids (aa) in length, and which resemble the fibronectin type III (FN III) and immunoglobulin (Ig) folds. Two conserved disulfide bridges are found in the N-terminal sub-domain, while a WSXWS (SEQ ID NO: 22) motif is characteristic for the C-terminal sub-domain. Both LR CRH modules are separated by an Ig-like domain, and are followed by two membrane proximal FN III domains (FIG. 1). Using a panel of deletion and substitution mutants, Fong and co-workers (1998) showed that the membrane proximal CRH domain is necessary and sufficient for leptin binding, and that the two FN III domains are not involved in ligand binding. Thus far, six isoforms of the LR generated by alternative mRNA splicing have been recognized and termed LRa through LRf. The LR long form (LRlo, or LRb) has an intracellular chain length of 302 aa, and is the only isoform capable of efficient signaling. It is this LRlo isoform that is primarily expressed in specific nuclei of the hypothalamus (Mercer et al., 1996; Fei et al., 1997; Schwartz et al., 1996), but expression at lower levels in other cell-types has also been observed (Hoggard et al., 1997; Ghilardi et al., 1996; Dyer et al., 1997). A second isoform, LRa, is a variant lacking most of the cytosolic domain. This LR short form (LRsh) is much more widely expressed, often at higher levels compared to LRlo, e.g., in the choroid plexus, kidney, lung, and liver (Tartaglia, 1997).
As activation of the leptin receptor by binding of leptin plays a role in several physiological processes, several variant and mutant forms of leptin and leptin receptors have been described that can be used to modulate leptin signaling. WO9605309 discloses, amongst others, antibodies against leptin. WO9812224 describes the use of fragments, derived from leptin, as leptin antagonist, especially for treating type II diabetes. The use of leptin antagonists is known to the person skilled in the art and includes, but is not limited to, diseases and conditions associated with obesity such as atherosclerosis, hypertension and type II diabetes, the modulation of body weight, the modulation of inflammation, the modulation of immune responses and autoimmune diseases.
Most modulators are based on preventing the interaction of leptin with the membrane-bound leptin receptor. However, it would be interesting to block leptin-induced signaling without blocking the interaction of leptin with the soluble receptor, as this would increase the flexibility of the regulation.
Surprisingly, we found that binding of a compound to non-leptin-binding domains of the extracellular part of the leptin receptor can block the leptin-induced signaling without blocking the leptin binding. This inhibitory effect is realized by disturbing the leptin-induced clustering of the receptor and the consequent signaling. Indeed, inhibiting the fibronectin III-fibronectin III domain interaction in the leptin receptor can block the leptin-induced signaling. This inhibition can be realized by a soluble fibronectin III domain of the leptin receptor and/or by a fibronectin III domain binding antibody. Alternatively, an antibody directed against the Ig-like domain of the leptin receptor may be used.